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Applied and Environmental Microbiology, August 2005, p. 4523-4530, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4523-4530.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR

Jed A. Fuhrman,1* Xiaolin Liang,1 and Rachel T. Noble2

Department of Biological Sciences and Wrigley Institute for Environmental Studies, University of Southern California, Los Angeles, California 90089-0371,1 Institute of Marine Sciences, The University of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City, North Carolina 285572

Received 14 July 2004/ Accepted 10 March 2005

Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r2 = 0.99) in fresh water and 23% (r2 = 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <104 enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r2 = 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r2 = 0.93; HA 12%, r2 = 0.87). The optimized method was used with 1-liter field samples from two very different freshwater "creeks" that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.

* Corresponding author. Mailing address: Department of Biological Sciences and Wrigley Institute for Environmental Studies, University of Southern California, Los Angeles, CA 90089-0371. Phone: (213) 740-5757. Fax: (213) 740-8123. E-mail: fuhrman{at}usc.edu.

Applied and Environmental Microbiology, August 2005, p. 4523-4530, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4523-4530.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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